Illumina DNA PCR-Free Prep, Tagmentation libraries require custom sequencing primers for successful sequencing on Illumina platforms because these libraries use novel chemistry. Failure to use the specified custom sequencing primers for these libraries results in sequencing failure on Illumina instruments. This bulletin reviews the custom sequencing primer kit contents, custom primer loading volume and position requirements per instrument, and how to set up runs using custom primers.

The custom sequencing primers, sold separately, are provided at final concentration and require no further dilution. They are loaded into the dedicated custom primer wells, rather than spiked into the Illumina primer wells. For full instructions, refer to the Custom Primers Guide for the respective instrument, listed in the “Resources” section of this bulletin.

These custom sequencing primers are compatible with Illumina DNA PCR-Free libraries, PhiX control library (Cat. No. FC-110-3001), and all currently supported/compatible Illumina libraries.

The required custom sequencing primers are available in two kit configurations, and are sold separately from the Illumina DNA PCR-Free library preparation kit:

• Illumina DNA PCR-Free Prep Sequencing Primers Read 1 (Cat. No. 20041796)
  • Contains VP10 Custom Read 1 Primer (4 tubes X 7.5 ml)
  • For use on the NovaSeq 6000 (SBS v1.0 only), MiSeq, and HiSeq 1000/1500/2000/2500
• Illumina DNA PCR-Free Prep Sequencing Primers Read 1 + Index 2 (Cat. No. 20041797)
  • Contains VP10 Custom Read 1 Primer (2 tubes X 7.5 ml) and VP14 Custom Index 2 Primer (2 tubes X 10.5 ml)
  • For use on iSeq 100, MiniSeq, NextSeq 500/550, NextSeq 1000/2000 and HiSeq 3000/4000

Custom primer loading volume and position requirements per instrument:

1. Requires iSeq 100 Control Software v3.0 or higher. Custom primers workflow must be enabled and primers added sequentially. See iSeq 100 Custom Primers guide for further instructions.
2. Requires NCS2k v1.2 or higher and v3 flow cells.
3. Primers required for sequencing Illumina DNA PCR-free Prep, Tagmentation libraries are included with NovaSeq v1.5 SBS cartridges, which require NovaSeq Control Software v1.7 or higher.

Run set up instructions per software:

1. Leave “Denature and Dilute Onboard” box checked. For more details on appropriate well selection, see the NextSeq 1000 and 2000 Custom Primers Guide.
2. Read length is limited by the number of SBS cycles available in reagent kits, maximum read length for Illumina sequencing platforms, and 10 bp dual indexes
3. IEM sample sheet creation is required only for MiSeq run setup. IEM can create sample sheets for demultiplexing purposes for other instruments 
4. Recommend run setup in Local Run Manager or standalone mode

Resources:

NovaSeq Series Custom Primers Guide
HiSeq System Custom Primers Guide
NextSeq 500 and NextSeq 550 Sequencing Systems Custom Primers Guide
NextSeq 1000/2000 Custom Primers Guide
MiSeq System Custom Primers Guide
MiniSeq System Custom Primers Guide
iSeq 100 Custom Primers Guide
Optimal loading concentrations for Illumina DNA PCR-Free libraries application note